Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.     

Purity assessment of recombinant human granulocyte colony‐stimulating factor in finished drug product by capillary zone electrophoresis

Current methods for determination of impurities with different charge‐to‐volume ratio are limited especially in terms of sensitivity and precision. The main goal of this research was to establish a quantitative method for determination of impurities with charges differing from that of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF, filgrastim) with superior precision and sensitivity compared to existing methods. A CZE method has been developed, optimized, and validated for a purity assessment of filgrastim in liquid pharmaceutical formulations. Optimal separation of filgrastim from the related impurities with different charges was achieved on a 50 μm id fused‐silica capillary of a total length of 80.5 cm. A BGE that contains 100 mM phosphoric acid adjusted to pH 7.0 with triethanolamine was used. The applied voltage was 20 kV while the temperature was maintained at 25°C. UV detection was set to 200 nm. Method was validated in terms of selectivity/specificity, linearity, precision, LOD, LOQ, stability, and robustness. Linearity was observed in the concentration range of 6–600 μg/mL and the LOQ was determined to be 0.3% relative to the concentration of filgrastim of 0.6 mg/mL. Other validation parameters were also found to be acceptable; thus the method was successfully applied for a quantitative purity assessment of filgrastim in a finished drug product.     

Validation of the anti-factor Xa assay for the potency assessment of enoxaparin in pharmaceutical formulations

Enoxaparin is a low-molecular weight heparin used clinically for the prevention and treatment of venous and arterial thrombosis. An anti-factor Xa assay was used to evaluate the potency of the final drug preparation. Method validation investigated parameters such as the range, linearity (r2 = 0.9971), precision, accuracy, and robustness; the biological assay incorporated a chromogenic endpoint and detection at 405 nm. The method yielded good results with a quantitation limit of 0.037 IU/mL and a detection limit of 0.011 IU/mL. The results demonstrated the validity of the anti-factor Xa assay for the determination of enoxaparin.     

A new evaluation technique for the detection of impurities in purified proteins via CE with native UV‐LIF

An analytical methodology for quality control analyses of IgG and their impurities is presented using a new UV‐LIF (266 nm) detector inside the cassette of a CE instrument and its performance was evaluated. The observed sensitivity was very close to that obtained by silver staining of slab gels (LOD of 25 ng/mL), while the sensitivity of the analysis is 80 times better than with CE/UV absorption (214 nm). Examples of the analysis of pharmaceutical and other commercial IgGs are provided and the kinetics of the reduction of IgG by β‐mercaptoethanol is reported, demonstrating the ease of performing the analysis.     

Enantioselective assay of chloroquine and its main metabolite deethyl chloroquine in human plasma by capillary electrophoresis

A sensitive assay for the determination of chloroquine (Clq) and its pharmacologically active metabolite deethyl chloroquine in plasma by capillary electrophoresis (CE) is developed. Plasma levels of drug and metabolite are measured using HeCd laser-induced fluorescence (LIF) detection over a range of three orders of magnitude from 2 to 1000 ng/mL after liquid-liquid extraction. A limit of detection of 0.5 ng/mL is achieved. Validation of the method yields intra- and interday precision data within the limits of 10% (20% at limit of quantitation) and intra- and interday accuracy data greater than 6% throughout the whole working range. The method is applied for the drug monitoring of patients treated with Clq. Based upon this assay, two enantioselective CE-LIF methods for Clq and its main metabolite are developed. Mixtures of substituted gamma-cyclodextrins are used as chiral selectors. A baseline separation of the enantiomers of both analytes in one run is achieved in less than 11 min (method A) and less than 9 min (method B), respectively. Hydroxychloroquine is used as the internal standard for both methods.     

Assay of urinary alpha-fluoro-beta-alanine by gas chromatography-mass spectrometry for the biological monitoring of occupational exposure to 5-fluorouracil in oncology nurses and pharmacy technicians

The validation of an analytical method for the measurement of the unnatural amino acid alpha-fluoro-beta-alanine (AFBA), the main metabolite of the antineoplastic drug 5-fluorouracil (5FU), in urine for the biological monitoring of the exposure of hospital workers to the drug when preparing the therapeutical doses and administering to cancer patients is described. The method employed a two-step extractive derivatization of the analyte from urine to the N-trifluoroacety-n-butyl ester derivative and detection by selected-ion monitoring gas chromatography-mass spectrometry of structurally specific fragments. The limit of detection was 20 ng/mL with quantification accuracy better than +/-20% and precision (CV%) better than +/-20% in the range 0.020-10 microg/mL. Norleucine was used as the internal standard and the sample-to-sample analysis time was less than 15 min. The validated method has been applied to the biological monitoring of some hospital workers potentially exposed to 5FU and to matched control subjects. On a total number of 65 analyzed urine samples from control and exposed subjects, only three, obtained from exposed subjects, were found to be positive, with values of 20, 30 and 1150 ng/mL, respectively.     

Determination of Cyanide in Blood for Forensic Toxicology Purposes—A Novel Nci Gc-Ms/Ms Technique

One of the recently evolving methods for cyanide determination in body fluids is GC-MS, following extractive alkylation with pentafluorobenzyl bromide or pentafluorobenzyl p-toluenesulfonate. The aim of this study was to improve previous GC methods by utilizing a triple quadrupole mass spectrometer, which could enhance selectivity and sensitivity allowing for the reliable confirmation of cyanide exposure in toxicological studies. Another purpose of this study was to facilitate a case investigation including a determination of cyanide in blood and to use the obtained data to confirm the ingestion of a substance, found together with a human corpse at the forensic scene. The blood samples were prepared following extractive alkylation with a phase transfer catalyst tetrabutylammonium sulfate and the PFB-Br derivatization agent. Optimal parameters for detection, including ionization type and multiple reaction monitoring (MRM) transitions had been investigated and then selected. The validation parameters for the above method were as follows—linear regression R² = 0.9997 in the range of 0.1 µg/mL to 10 µg/mL; LOD = 24 ng/mL; LOQ = 80 ng/mL and an average recovery of extraction of 98%. Our study demonstrates the first attempt of cyanide determination in blood with gas chromatography-tandem mass spectrometry. The established method could be applied in forensic studies due to MS/MS confirmation of organic cyanide derivative and low matrix interferences owning to utilizing negative chemical ionization.     

Validation of an oil-in-water microemulsion liquid chromatography method for analysis of perindopril tert-butylamine and its impurities

This paper describes the development and validation of a microemulsion liquid chromatography (MELC) method for simultaneous determination of perindopril tert-butylamine and its impurities in bulk active substances and the pharmaceutical dosage form of tablets. An appropriate resolution with reasonable retention times was obtained for a microemulsion containing 0.24% (w/v) butyl acetate, 0.30% (w/v) ethyl acetate, 2% (w/v) sodium dodecyl sulfate, 7.75% (w/v) n-butanol, and 20.0 mM potassium dihydrogen phosphate, the pH of which was adjusted to 3.70 with 85% orthophosphoric acid. Separations were performed on a Nucleosil 120-5 butyl modified (C4), 250 x 4 mm, 5 microm particle size silica column at 40 degrees C, with a mobile phase flow rate of 1.25 mL/min. UV detection was performed at 254 nm. The established method was subjected to method validation, and required validation parameters were defined. Robustness testing, an important part of method validation, was performed as well. Since robustness validation can be conducted using different experimental designs, the Plackett-Burman design was applied due to its possibility of testing many factors at the same time. The validated MELC method was found to be suitable for the simultaneous determination of perindopril tert-butylamine and its impurities in pharmaceuticals.     

Determination of tianeptine in tablets by high-performance liquid chromatography with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-CI). A mobile phase consisting of acetonitrile-10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45-300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.     

Development and validation of a capillary electrophoresis method with laser-induced fluorescence detection for the determination of captopril in human urine and pharmaceutical preparations

This study describes the development of a CE method for the analysis of the antihypertensive drug captopril using LIF detection. The method is based on the derivatization of captopril with the fluorescent label 5-iodoacetamidofluorescein. The optimization of the electrophoretic electrolyte composition together with other variables, such as applied voltage and injection time, resulted in a solution of 20 mM phosphate buffer adjusted to pH 12.0. The calibration curve for the fluorescent captopril derivative was linear in the concentration range 3.5-6000 ng/mL with a detection limit of 0.5 ng/mL. Intra- and interday precision (at a concentration of about 100 times the LOD) were less than 0.86 and 1.16%, respectively, both expressed as RSD. The assay was successfully used for quantification of captopril in some marketed pharmaceutical preparations and urine samples.     

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

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

High‐sensitivity simultaneous quantification of tacrolimus and 13‐O‐demethyl tacrolimus in human whole blood using ultra‐performance liquid chromatography coupled to tandem mass spectrometry

Blood concentrations of tacrolimus show large variability among patients and the narrow therapeutic range is related to adverse effects. Therefore, therapeutic drug monitoring is needed for strict management. 13‐O‐Demethyl tacrolimus (13‐O‐DMT) was reported as the major metabolite formed by cytochrome P450 (CYP)3A such as CYP3A5. In previous studies, the best lower limit of quantification (LLOQ) was 0.1 ng/mL for both substances. However, this LLOQ may not be low enough now because the dosage of tacrolimus has decreased in recent years. The purpose of this study was to develop and validate a high‐sensitivity and high‐throughput assay for simultaneous quantification of tacrolimus and 13‐O‐DMT in human whole blood using ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Thirty‐five stable kidney transplant recipients receiving tacrolimus were recruited in this study. The calibration curve range was 0.04–40 ng/mL. All calibration samples and quality control samples fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation. Trough concentrations of tacrolimus and 13‐O‐DMT in 35 stable kidney transplant recipients receiving tacrolimus were within the range of the respective calibration curve. Our novel UPLC–MS/MS method is more sensitive than previous methods for quantification of tacrolimus and 13‐O‐DMT.     

A Scientific, General Approach to Robustness Validation for an Ion-Pair LC Assay Method Through a Case Study

This paper describes a scientifically sound, systematic approach to the robustness validation of an ion-pair LC assay method. Robustness validation was carried out on all chromatographic parameters, such as ion-pair reagent concentration, buffer concentration and pH, organic to buffer ratio, column oven temperature, and column age. Small, deliberate variations to each parameter were implemented according to reasonably expected laboratory variabilities. For instance, a typical measuring variability with a 2,000-mL graduated cylinder is 20 mL, and this translates into variations of ±1.3% for a volume of 1,560 mL of an aqueous phase. Details on the experimental design of robustness validation are described, such as the parameters considered, the variations chosen for each parameter along with rationales, the probes that were used to assess robustness, and corroboration of method robustness with intermediate precision validation. This robustness validation approach eliminates guess work, ensures scientifically appropriate and customized variations for each method parameter, and has won regulatory acceptance.     

A validated HPLC assay to monitor riluzole plasma or serum concentrations in patients with amyotrophic lateral sclerosis

A specific, accurate and precise high-performance liquid chromatographic assay was developed for the determination of riluzole, a drug used to treat patients with amyotrophic lateral sclerosis. Samples were treated by extraction with dichloromethane followed by reversed-phase chromatography with ultraviolet detection at 260 nm. Preset validation criteria were met from 20 to 2000 ng/mL with a linear response curve. Extraction recovery of riluzole was 65-76%. The accuracy of the method was 102-103%. Intra- and inter-day coefficients of variation were in the ranges 2.8-4.9% and 1.8-9.7%. A detection limit of 5 ng/mL was found. Determination of concentrations in serum and plasma resulted in similar results below 500 ng/mL. At higher values a matrix effect cannot be excluded. This presented method can be used to monitor plasma or serum levels in ALS patients treated with riluzole.     

Development and validation of gas chromatography-mass spectroscopy method for determination of prilocaine HCl in human plasma using internal standard methodology

The accurate determination of prilocaine HCl levels in plasma is important in both clinical and pharmacological/toxicological studies. Prilocaine HCl is quickly hydrolyzed to o-toluidine, causing methemoglobinemia. For this, the present work describes the methodology and validation of a GC-MS assay for determination of prilocaine HCl with lidocaine HCl as internal standard in plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC-MS was 20-250 ng/mL in plasma. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 6.0%, and accuracy (relative error) was better than 9.0% (n = 6). The analytical recovery of prilocaine HCl and IS from plasma has averaged 94.79 and 96.8%, respectively. LOQ and LOD values for plasma were found to be 20 and 10 ng/mL, respectively. The GC-MS method can be used for determination from plasma of prilocaine HCl in routine measurement as well as in pharmacokinetic studies for clinical use.     

LC–MS/MS method development for quantification of doxorubicin and its metabolite 13‐hydroxy doxorubicin in mice biological matrices: Application to a pharmaco‐delivery study

This study describes the development of simple, rapid and sensitive liquid chromatography tandem mass spectrometry method for the simultaneous analysis of doxorubicin and its major metabolite, doxorubicinol, in mouse plasma, urine and tissues. The calibration curves were linear over the range 5–250 ng/mL for doxorubicin and 1.25–25 ng/mL for doxorubicinol in plasma and tumor, over the range 25–500 ng/mL for doxorubicin and 1.25–25 ng/mL for doxorubicinol in liver and kidney, and over the range 25–1000 ng/mL for doxorubicin and doxorubicinol in urine. The study was validated, using quality control samples prepared in all different matrices, for accuracy, precision, linearity, selectivity, lower limit of quantification and recovery in accordance with the US Food & Drug Administration guidelines. The method was successfully applied in determining the pharmaco‐distribution of doxorubicin and doxorubicinol after intravenously administration in tumor‐bearing mice of drug, free or nano‐formulated in ferritin nanoparticles or in liposomes. Obtained results demonstrate an effective different distribution and doxorubicin protection against metabolism linked to nano‐formulation. This method, thanks to its validation in plasma and urine, could be a powerful tool for pharmaceutical research and therapeutic drug monitoring, which is a clinical approach currently used in the optimization of oncologic treatments.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

A Novel Ultra-High Performance Liquid Chromatography Method for the Determination of Erdosteine,Related Impurities and Degradation Products in New Effervescent Tablets

A novel and automated, stability-indicating, reversed phase ultra performance liquid chromatography (UPLC) method was developed and validated for the quantitative determination of erdosteine, its known impurities and two novel degradation products in a new pharmaceutical dosage form (effervescent tablets). The chromatographic separations were performed on a Waters Acquity UPLC HSS T3, 1.8 µm (2.1 mm?×?150 mm, I.D.) stainless steel column. The mobile phase consisted of 0.1% TFA in water and methanol under gradient elution conditions, at a flow rate of 0.29 mL/min, for the assay and impurities analysis. UV detection was set at a wavelength of 238 nm. Erdosteine raw material, placebo and effervescent tablets were subjected to forced degradation. The new degradation products (labeled OX1 and OX2) were found after oxidative treatment and characterized by ultra performance liquid chromatography mass spectrometry. The validation parameters such as linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision, specificity and robustness were highly satisfactory for all analyzed compounds. LOD (0.020 and 0.011–0.385 µg/mL for erdosteine and impurities, respectively) and LOQ values show the high sensibility of the method. Specificity of the method was confirmed by testing the matrix components. The validated method demonstrated to be suitable for routine quality control purposes and for routine stability studies of erdosteine in effervescent formulations.     

Optimization of CE-SDS Method for Antibody Separation Based on Multi-Users Experimental Practices

Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) method with UV detection was developed and satisfactorily used for determination of purity and manufacturing consistency of a monoclonal antibody (MAb) at Amgen Inc. (Seattle, WA). When this method was applied to some other MAbs, several problems with method robustness became apparent. These issues resulted in abnormal Electropherogram (e-gram) profiles potentially linked to various parameters specific molecules analyzed, sample formulation buffer composition, CE-SDS gel matrix type, and operators. A multi-users interest group (called CE Users Forum) was formed to systematically investigate and understand these issues. The CE Users Forum first identified the issues which needed resolution, defined group experiments to better understand the problem and to test potential solutions, and together defined a generic (platform) CE-SDS method for MAbs. Two CE instruments, Agilent HP^3DCE and Beckman PA 800, two CE-SDS gel matrices, BioRad and Beckman gels, as well as different types of MAbs in various buffers were used in this investigation. We present here a platform CE-SDS method for purity determination of MAbs. Method optimization and trouble-shooting procedures by the CE Users Forum played a key role in delivering a robust analytical method for characterization of antibodies by improving instrumental and experimental parameters such as instrument variability, instrument operating parameters, operator training, and reagent stability. The optimized CE-SDS method is used during process development and has been transferred to the quality control (QC) lab as a purity assay for lot release testing of therapeutic antibodies. Any trained analyst can successfully perform this method. A group such as the CE Users Forum is a good way to integrate best practices and solve technical issues in a cooperative environment.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005     

Determination of N-methyl-4-isoleucine-cyclosporin (NIM811) in human whole blood by high performance liquid chromatography-tandem mass spectrometry

A liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of N-methyl-4-isoleucine-cyclosporin (NIM811) was developed and validated over the concentration range 1-2500 ng/mL in human whole blood using a 0.05 mL sample volume. NIM811 and the internal standard, d(12)-cyclosporin A (d(12)-CsA), were extracted from blood using MTBE via liquid-liquid extraction. After evaporation of the organic solvent and reconstitution, a 10 microL aliquot of the resulting extract was injected onto the LC-MS/MS system. Chromatographic separation of NIM811 and internal standard was performed using a Waters Symmetry RP-8 (50 x 4.6 mm, 3 microm particle size) column. The mobile phase consists of 10 mm ammonium acetate in water (A) and acetonitrile (B), with 45% B from 0 to 0.2 min, 45 to 85% B from 0.2 to 0.8 min and 85% B from 0.8 to 2.2 min. The total run time was 3.5 min with a flow rate of 0.8 mL/min. The method was validated for sensitivity, linearity, reproducibility, stability, dilution integrity and recovery. The precision and accuracy of quality control samples at low (2.00 ng/mL), medium (20.0 and 400 ng/mL) and high (2000 ng/mL) concentrations were in the range 1.1-4.3% relative standard deviation (RSD) and -2.5-10.0% (bias), respectively, from three validation runs. The method has been used to measure the exposure of NIM811 in human subjects.