UHPLC Method Research Articles

Quantitative determination of flavins – complex analyte/matrix effects in the presence of food colour additives

This paper reports a study on analyte/matrix effects of multicomponent FL mixtures of lumichrome (1), 3-methyl-lumiflavin (2), riboflavin (3) in the presence of uric acid (4), redox-stabilizers such as resorufin (5), resazurin (6) and naturally plant originating food color additive maclurin (7), respectively. Sampling complexity, in particular the spatial distribution of the experimental parameters, which can vary widely, is a critical challenge for achieving the selectivity for application to the environmental and foodstuff assays. The random distribution of the analyte in a real sample as well as the systematic and random errors at each of the analytical steps alter the variables of interest. The samples are very complex regarding the nature and matrix effects; significantly alert the recovery, than the choice of an instrumental method for determination, is the most important step. This paper places emphasis on UV-MALDI-Orbitrap-MS among the irreplaceable robust mass spectrometric methods. Despite its conceptually different quantitation procedure from the HPLC-ESI(or APCI)-MS ones, the method provided accurate and precise analytical information over a large concentration range up to 100 fg μL−1, high resolution power and screening ability of individual molecules in complex mixtures, solution, semi-liquid and solid-state analysis, short assay time, fast and step-limited sample pretreatment stages. The reported protocol is constructed on real analytical problems as an effective approach, ensuring maximal data representativeness towards the matrix effects typical for FL assay (pH scale ∈ 2.0–11.8, solvent polarity e ∈ 7.5–80.1, solvent donor number DN ∈ 14.1–31.5 kcal mol−1, T ∈ 25–300 °C), because of 1–7 stabilized different tautomeric forms, redox states and hydrogen bonding networks. The content included: (a) experimental and theoretical MS analyses and factors stabilizing the ionic fragments in the GP, determining the intensity; (b) full validated (methods and techniques) ESI-, APCI- and MALDI-MS protocols; (c) comparative analysis, employing EAs, LC, HPLC, and UHPLC methods and (d) chemometrics. The promising metrology achieved, beyond that of the reported UPLC-MS/MS, MW-HPLC, and UHPLC ones, evidenced the great capability of the UV-MALDI-Orbitrap-MS method for environmental and foodstuff monitoring versus the widely implemented microbial, chromatographic and spectrometric methods.

Development and validation of rapid UHPLC method for determination of aciclovir, its impurities and preservatives in topical cream

The aim of this study was to develop a new, rapid and sensitive method for simultaneous determination of aciclovir, three related substances and two preservatives methyl and propyl parahydroxybenzoates present in topical cream. This important active substance, aciclovir is the drug of the first choice for treatment of Herpes viral infections. Pharmacological mechanism is inhibition of replication of viral particles. Its major related substances are guanine, diguanine and diacetylaciclovir and they all were evaluated in this study. Preparation of the cream samples was very rapid and consisted of dissolution, sonication and filtration through membrane filter 0.22 μm. Innovative and expanding UHPLC method was chosen for proper analysis due to its excellent separation efficiency and resolution. Binary gradient flow was optimized to a C18 chromatographic column (100 mm × 3.5 mm, 1.7 μm). 0.1 M ammonium acetate buffer pH 5.5 and acetonitrile–water–tetrahydrofuran (90 : 6 : 4, v/v/v) were used as mobile phases in gradient mode with flow rate of 0.5 ml min−1. Separation temperature was increased to rarely used 60 °C. Wavelength of UV detector was set to 254 nm. This effective method was validated in accordance with ICH guidelines. Linearity, precision, accuracy, specificity, robustness, detection and quantitation limits were evaluated. The significance of the developed method is based on the analysis of important active substance aciclovir, its impurities and preservative in pharmaceutical preparation using UHPLC system with relatively high separation temperature which leads to radical analysis time shortening up to five times and solvent saving up to ten times comparing to the conventional HPLC system using 5 μm particle packed analytical column.

Application of UHPLC for the simultaneous analysis of free amino acids and biogenic amines in ripened acid-curd cheeses

A new UHPLC method for the simultaneous determination of amino acids and biogenic amines in a single run, and its first application to profile ripened acid-curd cheeses was presented. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC), 23 amino acids and 15 amines were separated in 9min only (12min total run time), and eluates monitored using their UV response at 249nm. Limits of detection (0.05-0.29mg/100g) and quantification (0.16-0.97mg/100g), repeatability for sample preparation (1.0-6.1% RSD) and method recoveries (83-120%) were found suitable for cheese analysis. In total, 47 acid-curd cheeses classified into sub-groups like cooked, Quargel-type or grey cheeses were analyzed for their free amino acid and amine (histamine, tyramine, putrescine, cadaverine, and tryptamine) contents, which (as expected) were highlighted by a great variability. Total free amino acid levels ranged between less than 100 and more than 4000mg/100g (median 567mg/100g), implying that for some cheeses less or not ripened/fresh quark was used for production or, in contrast, a higher degree of proteolysis had occurred. For the sum of biogenic amines, median concentration was determined at 7.0mg/100g, while only 5% of all cheeses had levels higher than 161.9mg/100g. Thus, the obtained results suggest quite acceptable biogenic amine levels for (mostly underrated) ripened acid-curd cheeses, although partly exceptional high concentrations (>250mg/100g) were indeed observed in individual samples.

Identification and Quantification of Phytochemical Composition and Anti-inflammatory and Radical Scavenging Properties of Methanolic Extracts of Chinese Propolis

Fifteen propolis samples collected from different regions of China were investigated and compared for their phytochemical composition and anti-inflammatory and radical scavenging properties. Eleven compounds including caffeic, p-coumaric, ferulic, isoferulic, and 3,4-dimethylcaffeic acids, pinobanksin, chrysin, pinocembrin, galangin, pinobanksin 3-acetate, and caffeic acid phenylethyl ester were quantified for the 15 propolis samples using a UHPLC method, whereas 38 compounds were identified by UPLC/Q-TOF-MS. The 15 propolis samples significantly differed in their total phenolic and total flavonoid contents, as well as their phytochemical profiles. The methanol extracts of propolis also showed significant anti-inflammatory effects in LPS-stimulated RAW 264.7 mouse macrophage cells at 10 μg propolis extract/mL concentration. Additionally, the propolis samples differed in their DPPH, ABTS cation, hydroxyl, and peroxide radical scavenging capacities and ferric reducing abilities. The results from this study may be used to improve the commercial production and consumption of Chinese propolis products.

Validation and clinical application of an UHPLC method for simultaneous analysis of total homocysteine and cysteine in human plasma

Several studies indicate that high levels of homocysteine (Hcy) and L-cysteine (L-Cys) are independent risk factors for cardiovascular disease. The validation and clinical application of an ultra HPLC method for analysis of Hcy and L-Cys is described. The reported method is simple, sensitive, rapid, precise, and less aggressive than other previously reported methods. The effect of the derivatization reaction time, pH, and organic solvent contents in the mobile phase are described and discussed. Optimized conditions resulted in excellent peak shapes. Results of method validation showed a good linearity (r(2) ≥ 0.993) over the investigated concentration ranges and were observed for both compounds. The LOD and LOQ were 0.05 μM and 0.15 μM for Hcy and 0.24 μM and 0.80 μM for L-Cys, respectively. Validation results proved that the method precision was good and the accuracy was satisfactory. This validated method was successfully applied in an epidemiological study to measure and compare the prevalence of Hcy and L-Cys high levels in plasma of Portuguese type 2 diabetic patients with and without angiopathy. The study results showed that prevalence of hyperhomocysteinemia and hypercysteinemia were at least two times higher in diabetic patients with angiopathy compared to diabetics without angiopathy.

Enantioselective ultra-high and high performance liquid chromatography: A comparative study of columns based on the Whelk-O1 selector

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150mm, 100mm, 75mm and 50mm columns, either 4.6 or 3.0mm ID. A 100mm×4.6mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated Hmin values of 3.53μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μinter) of 5.07mm/s, and Hmin of 4.26 and 4.17μm for the two enantiomers of acenaphthenol, at μinter of 4.85mm/s and 4.24mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150mm, 100mm, 75mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250mm×4.6mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50mm×3.0mm ID column was used as a compromise between reduced mobile phase consumption (less than 1mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods.

Immobilization of Rubia tinctorum L. suspension cultures and its effects on alizarin and purpurin accumulation and biomass production

The present study is investigating the immobilization of Rubia tinctorum L. suspension cultures. The effects of three inoculation volumes and three immobilization materials (loofa, sisal and jute) on fresh and dry weights of biomass as well as on alizarin and purpurin production were determined in this study. Two grams of four-week old callus tissue were transferred to liquid medium to establish suspension cultures. After four weeks, suspension cultures of R.tinctorum at concentration of 8 × 105 living cells/ml were immobilized with lignocellulosic materials and the cells were attached to all immobilization materials at the end of the first week and started to form aggregates on them. At the fourth week of these batch systems, biomass was measured approximately three times higher than the starting suspension cultures. The highest fresh weight was obtained (339.40 g/l) from sisal with ½ inoculation ratio. Immobilization materials and inoculation volumes had an effect on dry weights, and accordingly, the most effective combinations were jute with ¼ (J3) and ½ (J1) inoculation volumes with 7.86 and 7.82 g/l dry weights, respectively. Alizarin and purpurin contents of immobilized cells, analyzed with U-HPLC method, were 6.05 and 22.91 times higher than inoculated cells. All immobilization materials used in this study had no negative effect on to cells and biomass accumulation was enhanced. Concomitantly with rapid biomass increase, alizarin and purpurin production was ascended.

A Rapid UHPLC Method for the Simultaneous Determination of Drugs from Different Therapeutic Groups in Surface Water and Wastewater

A SPE-UHPLC-UV method for the determination of 13 pharmaceuticals from different therapeutic groups in surface water and wastewater samples was proposed. The following three solid-phase-extraction (SPE) columns with polymeric sorbents were used as a pre-concentration step: the Oasis HLB (average recoveries 93.8 %), the Nexus (84.0 %) and the Bond Elut ENV (88.3 %). A reverse-phase UHPLC with a C18e monolithic column and gradient elution program was used to obtain the best separations for all 13 drugs in short analysis time (3.4 min). The LOD range for determined drugs was 0.02–0.18 μg L−1, and the concentration range for drugs found in water samples was 0.06–0.90 μg L−1. The proposed method was used to analyze different water samples, mostly from rivers, and can be used as a monitoring tool for environmental pollution.

Highly sensitive fast determination of entecavir in rat urine by means of hydrophilic interaction chromatography–ultra-high-performance liquid chromatography–tandem mass spectrometry

Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC–UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC–MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1ml, 30mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C213 N15, which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87–114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).

Characterization of in vitro metabolic profiles of cinitapride obtained with liver microsomes of humans and various mammal species using UHPLC and chemometric methods for data analysis

An ultra-high performance liquid chromatographic method has been utilized to obtain metabolic profiles of cinitapride with liver microsomes of humans and various mammal species such as rats, mice, mini pigs, dogs, and monkeys. Metabolites have been generated by incubation of cinitapride in the presence of microsomes using nicotinamide adenine dinucleotide phosphate as a cofactor. Incubation times from 15 to 60 min have been assayed. Cinitapride and its metabolites have been separated by reversed-phase C(18) mode using ammonium formate aqueous solution (pH 6.5) and acetonitrile as the components of the mobile phase. Concentrations of metabolites in the incubated samples have resulted in an excellent source of multivariate data to be used to extract metabolic information. Statistic parameters and principal component analysis have been used to compare the in vitro metabolism of humans with the other species.

Determination of ganciclovir in human plasma by ultra performance liquid chromatography–UV detection

Implement a sensitive UHPLC method for the assay of ganciclovir in human plasma. We developed and validated a chromatographic method coupled to ultraviolet detection for quantification of ganciclovir, with a short run time using a small volume of human plasma. Comparison of system performance was made with respect to analysis time, efficiency and sensitivity. Correlation coefficients (r) of the calibration curves ranged from 0.999744 to 0.999784. Within-day and between-day imprecision and inaccuracy, specificity and recovery were also evaluated for validation. The method was precise and accurate and the retention time was 0.7 min. The calibration curves were linear between 0.5 and 30 μg/mL. There was a good correlation between HPLC and UHPLC techniques. We developed a method that is currently applied in a clinical study assessing GCV plasma concentration variability after ganciclovir and valganciclovir administration.

Stress Degradation Behavior of Abacavir Sulfate and Development of a Suitable Stability-Indicating UHPLC Method for the Determination of Abacavir, its Related Substances, and Degradation Products

A novel, stability-indicating UHPLC method was developed for the quantitative determination of Abacavir sulfate, its related substances, and forced degradation impurities in bulk drugs. The chromatographic separation was achieved on a Waters Acquity BEH C8, 50 mm × 2.1 mm, 1.7 μm particle size column with a mobile containing a gradient mixture of solution A (0.10 % v/v o-phosphoric acid in water) and solution B (0.10% v/v o-phosphoric acid in methanol). The flow rate was set at 0.40 mL/min and the run time was 6.0 min. The drug substance was subjected to the stress studies of hydrolysis, oxidation, photolysis, and thermal degradation. Abacavir sulfate was found to degrade significantly under acidic hydrolysis and oxidative stress conditions. The formed degradation products were reported and were well-resolved from Abacavir and its related substances. The mass balance was found to be satisfactory in all of the stress conditions, thus proving the stability-indicating capability of the method. The developed UHPLC method was validated to be in agreement with ICH requirements and found to be rapid, accurate, precise, linear, specific, and suitable for the quantitative determination of related substances and degradants in the bulk drug samples of Abacavir sulfate.

A Stability Indicating U-HPLC Method for Milnacipran in Bulk Drugs and Pharmaceutical Dosage Forms

The objective of the current study was to develop a validated, specific and stability-indicating reverse phase UHPLC method for the quantitative determination of Milnacipran and its related substances. The determination was done for active pharmaceutical ingredient and its pharmaceutical dosage forms in the presence of degradation products, and its process-related impurities. The drug was subjected to stress conditions of hydrolysis (acid and base), oxidation, pho- tolysis and thermal degradation per International Conference on Harmonization (ICH) prescribed stress conditions to show the stability-indicating power of the method. Significant degradation was observed during acid, base, oxidative and neutral stress hydrolysis. The chromatographic conditions were optimized using an impurity-spiked solution and the samples generated from forced degradation studies. In the developed UHPLC method, the resolution between Milnacipran and its process-related impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for Milnacipran and it’s all the five impurities. The chromatographic separation was achieved on a C18 stationary phase. The method employed a linear gradient elution and the detection wavelength was set at 220 nm. The mobile phase consists of buffer and acetonitrile delivered at a flow rate of 0.2 mL?min–1. Buffer consists a mixture of 10 mM Sodium dihydrogen phosphate monohydrate and 10 mM hexane sulfonate sodium salt, pH adjusted to 2.5 using ortho phosphoric acid. The mobile phase A consists of buffer and acetonitrile (950:50, v/v) and mobile phase B consists of acetonitrile. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

Quality by Design: Multidimensional exploration of the design space in high performance liquid chromatography method development for better robustness before validation

Robust HPLC separations lead to fewer analysis failures and better method transfer as well as providing an assurance of quality. This work presents the systematic development of an optimal, robust, fast UHPLC method for the simultaneous assay of two APIs of an eye drop sample and their impurities, in accordance with Quality by Design principles. Chromatography software is employed to effectively generate design spaces (Method Operable Design Regions), which are subsequently employed to determine the final method conditions and to evaluate robustness prior to validation.

Development and validation of ultra high performance liquid chromatography–mass spectrometry method for LBH589 in mouse plasma and tissues

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC™ BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/ z 349.42 → 157.95 and of tobramycin at 468.2 → 163. Calibration curves for the UHPLC method (0.0025–1 μg/mL for plasma and tissue homogenates, equivalent to 0.0357–14.2857 μg/g for tissue samples) showed a linear range of detector responses ( r > 0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from −2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 μg/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 μg/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice.

Identification, preparation and UHPLC determination of process-related impurity in zolmitriptan

A new impurity was detected and determined using gradient ion-pair UHPLC method with UV detection in zolmitriptan (ZOL). Using MS, NMR and IR study the impurity was identified as (4 S,4′ S)-4,4′-(2,2′-(4-(dimethylamino)butane-1,1-diyl)bis(3-(2-(dimethylamino) ethyl)-1 H-indole-5,2-diyl))bis(methylene)di(oxazolidin-2-one) (ZOL-dimer). The standard of ZOL-dimer was consequently prepared via organic synthesis followed by semipreparative HPLC purification. The UHPLC method was optimized in order to selectively detect and quantify other known and unknown process-related impurities and degradation products of ZOL as well. The presented method which was validated with respect to linearity, accuracy, precision and selectivity has an advantage of a very quick UHPLC chromatographic separation (less than 7 min including re-equilibration time) and therefore is highly suitable for routine analysis of related substances and stability studies of ZOL.

Sensitive Analysis of Commonly Used Artificial and Natural Sweeteners Including Stevia and Their Impurities and Degradation Products

Many recently commercialized sweeteners tend to have increased potency, reducing the amount of active ingredient added to beverages and other food products and often providing cost savings to the manufacturer. Using increased potency has also contributed to a need for sensitive analytical methods to quantify the active product and to detect low levels of breakdown products and impurities, which are required for quality and safety issues. Because compounds typically do not possess any chromophore, traditional HPLC-UV approaches are inappropriate. The work in this study describes several methods using HPLC with the Thermo Scientific Dionex CoronaTM Charged Aerosol Detector (CADTM) for the study of common natural sugars (fructose, glucose, turanose, saccharose, trehalose, maltose, melezitose, and raffinose); artificial sweeteners (sucralose, aspartame, saccharin, and acesulfame K); and newly introduced products containing stevia extracts (rebaudioside A and stevioside). These HPLC-CAD methods provide sensitivity at low levels (ng) with good reproducibility and accuracy, and correlation to the component concentrations. Stevia products were analyzed by both charged aerosol detection and UV. Charged aerosol detection showed a greater than fivefold improvement in sensitivity over UV for all major components. Finally, the UHPLC methods developed showed a decreased run time and an increased sensitivity for glucose, lactose, and sucrose. Typical limits of detection (LOD) were found to be <500 pg (on column) for glucose and other monoand disaccharides. HPLC-CAD is a very flexible approach to measuring sweeteners and overcomes many of the limitations of UV, refractive index (RI), LCMS, evaporative light scattering detection (ELSD), and HPLC-pulsed amperometric approaches. The HPLC-CAD platform can be used throughout the manufacturing process to ensure finished quality and batch-to-batch uniformity. Introduction The Dionex Corona CAD is a mass-sensitive universal detector for nonvolatile and some semivolatile analytes. Unlike absorbance, fluorescence, RI, or electrochemical detection, analyte response is independent of chemical Sensitive Analysis of Commonly Used Artificial and Natural Sweeteners Including Stevia and Their Impurities and Degradation Products Ian Acworth, Chris Crafts, and Bruce Bailey, Thermo Fisher Scientific, Chelmsford, MA, USA structure. Carbohydrates lack chromophores and the typical approaches used for their analysis may lack sensitivity, require derivatization, or cannot be used with gradient. The work in this study examines different HPLC-CAD methods for the analysis of honey sugars, artificial sweeteners, and other natural products including stevia extracts. Finally, a new UHPLC method for fast analysis of simple carbohydrates is presented. The Corona CAD system is easy to use and offers a simple, sensitive, reproducible, and direct approach for the routine analysis of natural and artificial sweeteners. Artificial Sweeteners Global Method HPLC Parameters Column: ACE®, C18 4.6 × 250 mm, 5 μm Mobile Phase: A) Deionized water B) Acetonitrile + 0.1% trifluoroacetic acid Gradient: 2 to 40% B over 25 min, 40 to 60% 25–30 min Flow Rate: 1.0 mL/min Inj. Volume: 50 μL Column Temp.: 30 °C Samples: 1.2 to 20 μg on column FIGURE 1. Chromatogram of artificial sweeteners. 26831 5 10 15 20 25 30 35 400 A ce su lfa m e K C yc la m at e S ac ch ar in S uc ra lo se A sp ar ta m e

Development of a UHPLC method for the assessment of the metabolic profile of cinitapride

An ultra high-performance liquid chromatographic method was developed to study the cinitapride metabolism. Metabolites were generated from the incubation of cinitapride with human liver microsomes. Cinitapride and its metabolites were separated by reversed-phase mode using a formate aqueous solution (pH 6.5) and acetonitrile as the components of the mobile phase. Chromatographic conditions, including the establishment of an elution gradient, were optimized for obtaining the maximum number of resolved components in the minimum analysis time. Experimental design and multicriteria decision-making strategies were utilized to facilitate the optimization of chromatographic conditions. Figures of merit were evaluated with cinitapride standards and incubated samples. Limits of detection are about 0.03 μmol/L, and repeatabilities are better than 0.06% for retention times and better than 3.5% for concentrations. The method was applied to characterize the in vitro cinitapride metabolism with human liver microsomes.

A fast ultra high pressure liquid chromatographic method for qualification and quantification of pharmaceutical combination preparations containing paracetamol, acetyl salicylic acid and/or antihistaminics

A fully validated UHPLC method for the identification and quantification of pharmaceutical preparations, containing paracetamol and/or acetyl salicylic acid, combined with anti-histaminics (phenylephrine, pheniramine maleate, diphenhydramine, promethazine) and/or other additives as quinine sulphate, caffeine or codein phosphate, was developed. The proposed method uses a Waters Acquity BEH C18 column (2 mm × 100 mm, 1.7 μm) with a gradient using an ammonium acetate buffer pH 4.0 as aqueous phase and methanol as organic modifier. The obtained method was fully validated based on its measurement uncertainty (accuracy profile) and robustness tests. Calibration lines for all components were linear within the studied ranges. The relative bias and the relative standard deviations for all components were respectively smaller than 1.5% and 2%, the β-expectation tolerance limits did not exceed the acceptance limits of 10% and the relative expanded uncertainties were smaller than 5% for all of the considered components. A UHPLC method was obtained for the identification and quantification of these kind of pharmaceutical preparations, which will significantly reduce analysis times and workload for the laboratories charged with the quality control of these preparations.

Comparison of UHPLC and HPLC Methods for The Assay of Prostanoids: “Are The Methods Equivalent in Terms of Accuracy and Precision?”

As new methods are developed to increase efficiency and higher analytical performance, it is necessary to evaluate their quality in comparison to standard methods. To understand how the analytical performance changes between methods, it is common to compare the validation parameters; sensitivity, linearity, accuracy and precision. Here, we compare an UHPLC-UV method to the HPLC-UV method (reference method) for the simultaneous determination of seven prostanoids. Though the basic chromatography theory is the same for HPLC and UHPLC, the instrumentation has been modified to accommodate higher pressures, lower flow rates and smaller sample size. The differences in analytical instrumentation and procedures can give rise to method inequivalencies. Our approach evaluates the UHPLC and HPLC methods and poses the question: are the methods equivalent? To answer this question a statistical comparison of the analytical performance and method parameters is necessary. Statistical comparisons were performed using the t-test, F-test, regression analyses (ordinary linear regression and Deming regression) and Bland-Altman analyses. Statistical comparison of the results, suggested that the precision (amount of variability) is different (p < 0.05) for the HPLC and UHPLC methods. Whereas, the accuracy (method bias and the means) is similar (p > 0.05) for 8-isoprostane, 11-dehydro TXB₂, PGE₂ PGF(2α), PGD₂ and 15-deoxy Δ¹²,¹⁴ PGJ₂. Ordinary linear regression shows that the methods are well correlated for all compounds. The Deming regression, which assumes error in both the methods, suggests the existence of a proportional and constant bias for 11-dehydro TXB₂ and only proportional bias for 8-isoprostane, PGF(2α), PGD₂ and 15-deoxy Δ(12,14) PGJ₂ between the two methods. According to Deming regression, the two methods are statistically similar for 6-keto PGF(1α) and PGE₂. The Bland-Altman analyses indicate the two methods are commutable.