Endogenous Serum Components Research Articles

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Innovative Use of LC-MS/MS for Simultaneous Quantitation of Neutralizing Antibody, Residual Drug, and Human Immunoglobulin G in Immunogenicity Assay Development

Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays. However, in the BEAD, certain amounts of the drug and endogenous serum components (so-called residual drug and serum components) from serum samples are carried over to final BEAD eluates due to formation of protein complexes with ADA or nonspecific binding with the beads. Using current enzyme-linked immunosorbent assay (ELISA)-based ligand-binding assays, it is difficult to evaluate the residual drug, which is complexed with excessive amounts of ADA and endogenous serum components in the BEAD eluates. Here, we describe an innovative application of LC-MS/MS for simultaneous detection of the residual human monoclonal antibody drug and endogenous human IgG and the neutralizing antibody positive-control (NAb-PC) in the BEAD eluates. In this study, the low levels of the residual drug and human IgG in the BEAD eluates indicate that the BEAD efficiently removed the high-concentration drug and serum components from the serum samples. Meanwhile, the NAb-PC recovery (∼42%) in the BEAD provided an acceptable detection limit for the cell-based assay. This novel application of LC-MS/MS to immunogenicity assay development demonstrates the advantages of LC-MS/MS in selectivity and multiplexing, which provides direct and fast measurements of multiple components for immunogenicity assay development.

Monitoring of Pregabalin in Pharmaceutical Formulations and Human Serum Using UV and RP-HPLC Techniques: Application to Dissolution Test Method

Pregabalin an antiepileptic was the first compound approved by FDA for treating chronic pain. A sensitive, efficient, economical, environmental friendly and isocratic liquid chromatographic method for the determination of pregabalin in bulk drug, pharmaceutical dosage forms and human serum has been developed and validated according to ICH guidelines. Chromatographic separation was performed on a KROMASIL® 100-5 C-18 column (250×4.6 i.d. mm) (5 μm particle size) as stationary phase with a UV detection at 210 nm using isocratic elution when buffer pH 7 and acetonitrile (96:4, v/v) were used as the mobile phase and the flow rate was 1 ml min-1 at ambient temperature,the retention time was 4.6 minutes.The method showed good linearity in the range 1-25 μg mL-1 with R2>0.999. The lower limit of detection (LLOD) and quantitation (LLOQ) were 10 ng mL-1 and 17 ng mL-1 and 0.04 and 0.12 ng mL-1 for drug and serum, respectively. Validation of the method showed good precision and accuracy for the proposed method. The newly developed method can be successfully applied for the determination of pregabalin in active pharmaceutical ingredients, pharmaceutical formulations, human serum and could be used in therapeutic drug monitoring and clinical laboratories without diode array detector and without interference of excipients or endogenous components of serum.

Liquid chromatographic method for the simultaneous determination of captopril, piroxicam, and amlodipine in bulk drug, pharmaceutical formulation, and human serum by programming the detector

A highly sensitive LC method with UV detection has been developed for the simultaneous determination of coadministered drugs captopril, piroxicam, and amlodipine in bulk drug, pharmaceutical formulations, and human serum at the isosbestic point (235 nm) and at individual λmax (220, 255, and 238 nm, respectively) by programming the detector with time to match the individual analyte's chromophore, which enhanced the sensitivity with linear range. The assay involved an isocratic elution of analytes on a Bondapak C18 (10 μm, 25 × 0.46 cm) column at ambient temperature using a mobile phase of methanol/water 80:20 at pH 2.9 and a flow rate of 1.0 mL/min. Linearity was found to be 0.25-25, 0.10-6.0, and 0.20-13.0 μg/mL with correlation coefficient >0.998 and detection limits of 7.39, 3.90, and 9.38 ng/mL, respectively, whereas calibration curves for wavelength-programmed analysis were 0.10-6.0, 0.04-2.56, and 0.10-10.0 μg/mL with correlation coefficient >0.998 and detection limits of 5.79, 2.68, and 3.87 ng/mL, respectively. All the validated parameters were in the acceptable range. The recovery of drugs was 99.32-100.39 and 98.65-101.96% in pharmaceutical formulation and human serum, respectively, at the isosbestic point and at individual λmax . This method is applicable for the analysis of drugs in bulk drug, tablets, serum, and in clinical samples without interference of excipients or endogenous serum components.

An Ultra-sensitive LC Method for the Simultaneous Determination of Paracetamol, Carbamazepine, Losartan and Ciprofloxacin in Bulk Drug, Pharmaceutical Formulation and Human Serum by Programming the Detector

An ultra-sensitive LC method for the simultaneous quantitation of paracetamol, carbamazepine, losartan and ciprofloxacin have been developed and validated following the ICH guidelines at isobestic point and by programming the detector at individual wavelength of each component. The components were eluted by 50:50 v/v acetonitrile-water (pH 3.0) using a Bondapak, C18 (10 μm, 25 × 0.46 cm) column at flow rate of 1.0 mL·min-1 with detection wavelength 240 nm at isobestic point and 245, 230, 206 and 272 nm for paracetamol, carbamazepine, losartan potassium and ciprofloxacin respectively by programming the detector. Linearity was found to be 0.5 - 24, 0.25 - 8.0, 0.4 - 12 and 0.75 - 10 μg·mL-1 (R2 > 0.999) with detection limits 99, 20, 30 and 6.0 ng·mL-1 respectively. Comparison study with time program method showed more sensitivity with calibration range of 0.4 - 12, 0.2 - 6.0, 0.1 - 3.0 and 0.25 - 8.0 μg·mL-1 (R2 > 0.999) and LOD values 29, 11, 2.0 and 5.0 ng·mL-1 respectively. Percent recoveries >98.37% from pharmaceutical formulation and human serum samples and RSD 2% for inter-day and intra-day assay were obtained. The method was found to be robust and can be successfully applied for the determination of studied drugs in, pharmaceutical formulations and human serum without interference of excipients or endogenous components of serum.

An Ultra-Sensitive and Selective LC-UV Method for the Simultaneous Determination of Pravastatin, Diltiazem, Naproxen Sodium and Meloxicam in API, Pharmaceutical Formulations and Human Serum

The aim of the present work was to develop a sensitive liquid chromatographic method for the quantitation of pravastatin and diltiazem along with naproxen and meloxicam using ultraviolet detector. The separation of components was achieved on Purospher Star, C18 (5 µm, 25 x 0.46 cm) column using methanol-water (80:20 v/v) as mobile phase and pH adjusted 3.4 with 85% o-phosphoric acid. Related parameters that may influence the enrichment efficiency of speration of drugs such as the kind and volume of elute, sample flow rate, sample pH, and volume of the drug samples were investi-gated. Detection was performed at ambient temperature at 220 nm by pumping the mobile phase at the flow rate 1.0 mL min-1. The experimental results indicated a good linearity (R 2 > 0.9947) over the concentration range of 0.5-20 µg mL-1 for pravastatin, naproxen and meloxicam and 0.75-24 µg mL-1 for diltiazem. The method was compared by programming the detector adjusting the wavelength with time to match the individual analyte's chromophore which enhanced sensitivity with linear range 0.25-8.0, 0.5-16, 0.4-12 and 0.2-4.0 µg mL-1 for pravastatin, diltiazem, naproxen and meloxicam respectively. The LOD values shifted down from 33, 70, 50 and 80 ng mL-1 to 15, 42, 20 and 10 ng mL-1 for pravastatin, diltiazem, na-proxen and meloxicam respectively. Validation of the method showed good precision and accuracy for the proposed method. All the results indicated that this procedure could allow the simultaneous determination of these four compounds in API, pharmaceutical formulations and serum at trace levels. The method can be successfully applied for the determination of these drugs in human serum, clinical laboratories and in pharmaceutical formulations without diode array detector and without interference of excipients or endogenous components of serum.

Ultra Sensitive Liquid Chromatographic Method for the Simultaneous Determination of Carbamazepine with Nsaids in API, Pharmaceutical Formulation and Human Serum by Programming the Detector: Application to In Vitro Drug Interaction

Here we describe an ultra-sensitive reverse phase liquid chromatographic method for the simultaneous determination of carbamazepine with NSAIDs in API, pharmaceutical formulation and human serum with UV detection. The separation of these multiple drug components was made by a Bondapak C18 column (10 µm, 25 cm×0.46 cm) using a moderate acidic mixture of mobile phase i.e., methanol:water 80:20 with pH adjusted to 3.0 using 85% o-phosphoric acid. The flow rate of the mobile phase was affixed at 1.0 ml min -1 for the best minimum interval of elusion of all the analytes. The detector response was monitored at isobestic point of 220 nm at ambient temperature. Calibration curves were obtained in the range of 0.4-12 µg mL -1 for carbamazepine, 0.5-16 µg mL -1 for meloxicam and 0.25-8.0 µg mL -1 for ibuprofen and mefenamic acid. Limits of detection were found to be 4.0, 3.0, 1.0, and 13.0 ng mL-1 respectively. The method was compared by programming the detector at individual wavelength of components which showed more sensitivity with linear range 0.10-3.0, 0.15-5.0, 0.10-3.0 and 0.125-4.0 µg mL -1 and detection limits 2.0, 2.0, 1.0, and 3.0 ng mL-1 respectively. ICH guidelines were followed for validation study. The method showed good precision and accuracy within acceptable range and can be successfully applied for the determination of these drugs in pharmaceutical formulations, human serum and clinical laboratories without interference of excipients or endogenous components of serum. In vitro interaction studies have also been carried out at physiological temperature in buffers of various pH (4, 7.4 and 9.0) simulating human stomach environments and % availability has been calculated. Moreover, the solid charge transfer complexes of carbamazepine with interacting NSAIDs were synthesized and characterized by IR spectroscopy and verified by computational molecular modeling.

An Ultra-Sensitive LC Method for Simultaneous Determination of Rosuvastatin, Alprazolam and Diclofenac Sodium in API, Pharmaceutical Formulations and Human Serum by Programming the Detector

Here, we report a rapid and efficient liquid chromatographic method with UV detection for the simultaneous determination of rosuvastatin, alprazolam and diclofenac sodium in API, pharmaceutical formulations and human serum employing a reversed phase Bondapak C18 (25 cm, 0.46 cm, 10 μm) column at room temperature using methanol: water (80:20 v/v) and pH adjusted to 2.9 with 85% o-phosphoric acid. Separation was achieved within a time span of 6 min. The detector response was monitored at 240 nm and the flow rate was set at 1.0 mL.min-1. Various LC parameters were optimized and developed method was validated as per ICH (2006) guidelines. Linear calibration range was determined to be 0.02-0.64, 0.125-4.0 and 0.05-1.6 μg.mL-1 and detection and quantitation limits were found to be 4.0, 17.0, 7.0 and 12.0, 52.0, 22.0 ng.mL-1 for rosuvastatin, alprazolam and diclofenic sodium respectively. Method was linear for all analytes with correlation coefficients >0.998. The intra-day and inter-day accuracy and precision of the assay were in acceptable range of 98.21-101.91% recovery and 0.20-2.07% RSD. The sensitivity of the method was enhanced when the chromatograms were analyzed by programming the detector at individual λmax of 244, 222 and 284 nm for rosuvastatin, alprazolam and diclofenac sodium respectively. High peak intensity was observed on injecting same sample concentration after the detector was programmed. Linearity was obtained even at lower concentrations of 0.02-0.64, 0.05-1.60 and 0.02-0.64 μg.mL-1. LOD and LOQ values shifted down to 3.0, 6.0, 1.0 and 9.0, 19.0, 3.0 ng.mL-1 respectively. Recovery was found to be in the range of 98.32-101.87% and inter-day and intra-day precision was less than 2.10%. The proposed method was found to be robust and successfully employed for the determination of studied drugs in commercial formulations and human serum without interference of excipients or endogenous components of serum.

Determination of the active metabolites of sibutramine in rat serum using column‐switching HPLC

A simple and direct analysis using column-switching HPLC method was developed and validated for the quantification of active metabolites of sibutramine, N-mono-desmethyl metabolite (metabolite 1, M1) and N-di-desmethyl metabolite (metabolite 2, M2) in the serum of rats administered sibutramine HCl (5.0 mg/kg, p.o.). Rat serum was directly injected onto the precolumn without sample prepreparation step following dilution with mobile phase A, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (8.3:4.5:87.2 by volume). After the endogenous serum components were eluted to waste, the system was switched and the analytes were eluted to the trap column. Active metabolites M1 and M2 were then back-flushed to the analytical column for separation with mobile phase B, i. e., methanol-ACN-20 mM ammonium phosphate buffer (pH 6.0 with phosphoric acid) (35.8:19.2:45 by volume) and detected at 223 nm. The calibration curves of active metabolites M1 and M2 were linear in the range of 0.1-1.0 microg/mL and 0.15-1.8 microg/mL. This method was fully validated and shown to be specific, accurate (10.4-10.7% error), and precise (1.97-8.79% CV). This simple and rapid analytical method using column-switching appears to be useful for the pharmacokinetic study of active metabolites (M1 and M2) of sibutramine.

Simple and rapid assay for acetaminophen and conjugated metabolites in low-volume serum samples

The use of marker compounds for estimating drug metabolic capacity or pharmacokinetic parameters is common in the biological sciences. Often small laboratory animals are used and thus sample size is a limiting concern. In this report, we describe an assay we developed for measuring the concentration of acetaminophen and its conjugated metabolites in low-volume serum samples. Acetaminophen and metabolites were removed from 10 μl serum samples by a single-step 6% (v/v) perchloric acid deproteination using theophylline as internal standard. Samples were separated in a pH 2.2 sodium sulfate–acetonitrile mobile phase at a flow-rate of 1.5 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 254 nm. The resulting chromatograms showed no interfering peaks from endogenous serum components. The concentration ranges measured were 1.56–200 μg/ml for acetaminophen and acetaminophen sulfate and 3.91–500 μg/ml for acetaminophen glucuronide. The assay was linear in the range of concentrations analyzed. The intra-day and inter-day coefficient of variation ranged from 0.4 to 8.2% and 0.2 to 12.3% for acetaminophen, 0.5 to 12.9% and 0.3 to 16.1% for acetaminophen glucuronide, and 0.4 to 8.1% and 0.2 to 14.3% for acetaminophen sulfate, respectively. Results from the experiments show that acetaminophen and its conjugated metabolites can easily and reproducibly be measured in low-volume serum samples and thus may offer an additional method to measure these compounds when the volume of biological samples may be limited.

Gas chromatographic-mass spectrometric identification and quantitation of ethylene glycol in serum after derivatization with perfluorooctanoyl chloride: a novel derivative.

Ethylene glycol poisoning is a common clinical problem and identification as well as quantitation of ethylene glycol in serum is important for medical and legal purposes. Most investigators described determination of ethylene glycol by gas chromatography without derivatization or derivatives forming a molecular ion < 200. We describe a novel derivatization technique of ethylene glycol using perfluorooctanoyl chloride, after extraction from serum using acetone. This derivative has a molecular mass of 854 and produces a base peak at m/z 441 and other diagnostic strong peaks for unambiguous identification. Moreover, this derivative is less volatile and is free from interferences from endogenous serum components. Quantitation can be achieved by using 1,4-butanediol as an internal standard. The assay showed within-run and between-run precision of 7.2% and 8.0%, respectively, and linearity over the serum ethylene glycol concentration range 70-2240 micrograms/ml with a detection limit of 5 micrograms/ml.

Early blood-brain barrier disruption after high-dose single-fraction irradiation in rats.

We studied the effect of high-dose single-fraction irradiation on the permeability of the blood-brain barrier (BBB) in rat brains. Immunohistochemistry with an antibody to serum albumin was used as a sensitive method for detecting the extravasation of endogenous serum components. Extravasation of albumin was detected as early as 1 day after irradiation with 20 or 40 Gy. Immunoreactivity reached its maximum after 3 days, gradually decreased during the following few weeks and had disappeared by day 30. Extravasation was much greater after irradiation with 80 Gy and continued to increase during the whole period of the experiment (6 days). Disruption of BBB this early after irradiation has not been previously documented. The time course of observed serum albumin extravasation, however, agrees well with the previous ultrastructural evidence for increased BBB permeability after irradiation with 27 Gy in monkey brains. This transient impairment of BBB may contribute to the reversible neurological symptoms after radiosurgery. It may also allow drugs that normally not pass the BBB to do so and thus disperse in the brain when administered at this time.

Simultaneous quantification of zidovudine and its glucuronide in serum by high-performance liquid chromatography

A sensitive high-performance liquid chromatography (HPLC) assay has been developed to simultaneously determine levels of the anti human immunodeficiency virus agent, zidovudine (AZT), and its major metabolite (the 5′-O-glucuronide) in serum. Samples were first mixed with an internal standard (a stereisomer of AZT), then prepared for analysis using solid-phase extraction columns and chromatographed using a reversed-phase analytical column. Isocratic elution with a mobile phase of 15% acetonitrile, buffered to pH 2.70 with ammonium phosphate, gave good resolution of the three analytes and endogenous serum components. The HPLC analysis time required per sample was 34 min and analyte recoveries were reproducibly high (greater than 93%). Replicate analyses of prepared standards gave satisfactory precision and accuracy, with coefficients of variation less than 15% and deviations from expected concentrations less than 10%.

Quantitative Determination of Uric Acid in Serum by Reversed-Phase Liquid Chromatography Using an Internal Standard

Abstract A rapid and sensitive high-performance liquid chromatography procedure is reported for the analysis of uric acid in serum. The method employs a single treatment of serum with cyclohexane and an excess of ammonium sulfate to eliminate endogenous serum components which easily contaminate the chromatographic system. Reversed-phase chromatography is performed at room temperature using a binary-solvent gradient, monitoring the effluent at 290 nm. Quantitation is based on peak-height ratio of uric acid to internal standard (β-hydroxyethyltheophylline). A linear response is obtained to 160 mg/1. Within-day and between-day precisions for the mean of this range varied from 0.59 to 1.1% and from 0.71 to 1.2%, respectively. Numerous potential interfering substances tested do not interfere.

Determination of adriamycin, adriamycinol and their 7-deoxy aglycones in human serum by high-performance liquid chromatography

A reversed-phase isocratic high-performance liquid chromatographic assay is described for the measurement of adriamycin, adriamycinol and their 7-deoxyaglycones in human serum. The lower limit of detection in serum is 3 ng/ml for adriamycin and 1 ng/ml for adriamycinol and the 7-deoxyaglycones with coefficients of variation for k′ of less than 5% throughout the day. An extraction technique for serum is described which is capable of an almost equal recovery (>77%) of adriamycin, metabolites and daunorubicin (the internal standard) without interference from endogenous components of serum. Serum concentrations of metabolites 15 min after intravenous bolus administration of 40 mg/m 2 adriamycin in two different patients were 26.5 and 16.6 ng/ml for adriamycinol; 109.8 and 5.8 ng/ml for the adriamycinol 7-deoxyaglycone and 21.4 and 17.1 ng/ml for the adriamycin 7-deoxy- aglycone. A total of six metabolites of adriamycin were detected in the two patients using this methodology.