Analytical quality-by-design (AQbD) approach for comprehensive analysis of bioactive compounds from Citrus peel wastes by UPLC.

The growing interest in ingredients from natural sources has expanded the need for quality assessments of plant extracts. Analytical quality-by-design (AQbD) has been increasingly applied in regulated environments such as pharmaceutical industries and, more recently, for the bioactive compounds found in botanical materials. This work aimed to obtain qualitative (overall resolution and maximum peak capacity) and quantitative performances for target analytes using AQbD principles. The analytical target profile was elaborated; critical method parameters (independent variables) that affect the critical method attributes (dependent variables) were selected from a risk assessment for a reversed-phase liquid chromatography with diode array detection (RPLC-DAD) method. YMC-Triart C18 (3.0 × 100 mm, 1.9 µm) and a gradient elution using 0.2% acetic acid and methanol:acetonitrile 1:3 (v/v) were chosen as the stationary and mobile phases, respectively. The optimal and robust conditions (temperature at 33.3 °C, flow rate of 0.68 mL.min-1, and a gradient slope of 4.18%.min-1) were established by the method operable design region (MODR). The validation was performed by accuracy profiles using 90% expectation tolerance intervals for the selected compounds found in Citrus spp. using C. japonica as blank matrix. The lower limits of quantification for hesperidin, bergapten, herniarin, and citropten were 5.32, 0.40, 0.49, and 0.52 mg.L-1, respectively (acceptance limit was set at ± 20%). Nobiletin did not show an adequate quantitative performance.

Quantitation of certolizumab pegol by validated liquid chromatography methods

Abstract Certolizumab pegol (CZP) is a Fab' fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn's disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.

Development and validation of a rapid reversed-phase liquid chromatography method for CnAMP1 peptide quantification in human intestinal cell lines.

Plant foods are rich sources of biologically active peptides that may have a role in the prevention of diseases. Coconut water is a valuable beverage due to its nutrient composition and the presence of bioactive compounds, such as the peptide CnAMP1. It is unknown if CnAMP1 can be absorbed into intestinal cells. We, therefore, aimed to develop and validate a simple reversed-phase liquid chromatographic method to quantify the peptide in Caco-2 and LS180 cell lysates. CnAMP1 standards (1-200 µmol/L) and spiked cell lysates were injected onto a Reprosil-Pur 120 C18-AQ column (4.6 × 250 mm) using acetonitrile:water:trifluoroacetic acid (14.0:85.9:0.1, by volume) as mobile phase in isocratic mode at flow rate of 1 mL/min. The method achieved rapid separation (total run time of 6 min), with linear response, good sensitivity (limit of detection, 8.2 ng; lower limit of quantification, 30.6 ng) and no interfering peaks. Best recoveries (84-96%), accuracies (7.6-14.8%) and precision (1.5-8%) were found for LS180 cell lysates spiked with medium (50 µmol/L) and high (100 µmol/L) amounts of the peptide. Uptake assays detected no peptides in the cell lysates; however, after the first 15-min incubation CnAMP1 underwent partial hydrolysis upon incubation with LS180 cells (29%) and extensive hydrolysis with Caco-2 cells (93%).

Evaluation of recombinant human interferon beta 1b by liquid chromatography methods and bioassay

Recombinant human interferon beta 1b (rhIFNß-1b) is clinically used to treat multiple sclerosis. A reversed-phase liquid chromatography (RP-LC) method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was acetonitrile with 0.1% TFA run at a flow rate of 1.0 mL/min. A size exclusion liquid chromatography (SE-LC) method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.). The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM sodium phosphate dibasic and 200 mM sodium chloride buffer pH 7.4, run isocratically at a flow rate of 0.8 mL/min. Retention times were 31.87 and 17.78 min, and calibration curves were linear over the concentration range of 1-200 µg/mL (r2 = 0.9998) and 0.50-200 µg/mL (r2 = 0.9999), respectively, for RP-LC and SE-LC, with detection at 214 nm. Liquid chromatography (LC) methods were validated and employed in conjunction with the in vitro bioassay to assess the content/potency of rhIFNß-1b, contributing to improve the quality control and to ensure the efficacy of the biotherapeutic

Chiral separation of aryloxyphenoxy-propionate herbicides in a permethyl-ß-cyclodextrin based column. Influence of temperature and mobile phase composition on enantioselectivity.

We used a permethyl-ß-cyclodextrin chiral stationary phase under reversed-phase conditions for the chiral separation of four aryloxyphenoxy-propionate herbicides (fenoxaprop-p-ethyl, quizalofop-p-ethyl and tefuryl, and haloxyfop-p-methyl) with mixtures of methanol, ethanol, 2-propanol, n-propanol, tert-butanol, or acetonitrile and water as mobile phases and investigated the influence of mobile phase composition and column temperature (from 0 to 50°C) on the separation. The retention factors (k) and selectivity factors (α) of all the herbicides investigated decreased with increasing temperature. The lnα versus 1/T and lnk versus 1/T plots for the enantiomers of the chiral pesticides were linear within the range of 0-50°C with all alcohol/water mixtures constituting the mobile phase, but the lnk versus 1/T plots were nonlinear for all the enantiomers chromatographed in acetonitrile/water mixtures. The thermodynamic parameters based on linear van't Hoff plots were calculated. The influence of temperature and mobile phase composition on the enantioseparation of the solutes has rarely been considered simultaneously. The temperature and the solvents used in the mobile phase, however, were found to have a profound effect on the enantioseparation of these herbicides.

Analysis of streptokinase by validated liquid chromatography methods and correlation with an in vitro bioassay.

Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4 column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 µg/mL (25.75-25 750 IU/mL) (r2 = 0.9997) and 5-80 µg/mL (515-8240 IU/mL) (r2 = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.

Stability-indicating capillary zone electrophoresis method for the assessment of recombinant human interleukin-11 and its correlation with reversed-phase liquid chromatography and biossay.

A stability-indicating capillary zone electrophoresis (CZE) method was validated for the analysis of recombinant human interleukin-11(rhIL-11) using rupatadine fumarate, as internal standard (IS). A fused-silica capillary, (50 µm i.d.; effective length, 40 cm) was used at 25°C; the applied voltage was 20 kV. The background electrolyte solution consisted of 50 mmol L(-1) sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45 s, with detection by photodiode array detector set at 196 nm. Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The method was linear over the concentration range of 1.0-300 µg mL(-1) (r(2)=0.9992) and the limit of detection (LOD) and limit of quantitation (LOQ) were 0.2 µg mL(-1) and 1.0 µg mL(-1), respectively. The accuracy was 100.4% with bias lower than 1.1%. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p<0.05). The method was applied for the content/potency assessment of rhIL-11 in biopharmaceutical formulations, and the results were correlated to those of a validated reversed-phase LC method (RP-LC) and an TF-1 cell culture assay, showing non-significant differences (p>0.05). In addition the CZE and RP-LC methods were applied for the analysis of rhIL-11 in human plasma. Therefore, the proposed alternative method can be applied to monitor stability, to assure the batch-to-batch consistency and quality of the bulk and finished biotechnology-derived medicine.